Multiple myeloma (MM) is a common hematological cancer that with standard treatment is rarely curable. Although myeloma patients treated with immunomodulatory agents and or chemotherapy may experience a complete clinical response, the majority will eventually relapse. More aggressive treatments including allogeneic stem cell transplantation may be curative, but the high morbidity and mortality of myeloablative conditioning precludes use in many patients. The objective of this project is to develop a preclinical model of targeting myeloma stem cells with immunotherapy that in the long-term can be developed into a novel clinical therapeutic strategy that will improve outcomes for patients with myeloma. We and others have previously described a population of myeloma stem cells that possess clonogenic potential, are highly resistant to chemotheraputics agents, and can initiate tumorigenesis. One of the mechanisms that stem cells use to limit their exposure to chemotherapy is to express transport proteins that can pump out drug. This property of stem cells can be exploited for their purification, since the same transporters efflux Hoechst dye forming a distinct side population on flow cytometric analysis. These cells are likely to play a key role in disease relapse. We now propose to target these myeloma stem cells with immunotherapy directed against cancer testis antigens (CTAs). CTAs are expressed aberrantly in multiple myeloma, and by virtue of their immunogenecity and restricted expression in normal tissue such that T-cell tolerance does not develop, makes CTAs a promising antigen target in myeloma. Additionally, it was recently reported that some CTAs including NRAGE, NY-ESO, MAGE-1, and SSX are expressed in human mesenchymal stem cells (MSCs). This suggests that CTA expression may not only be a hallmark of gametogenesis but also a stem cell marker. Based on these observations, we hypothesize that myeloma stem cells preferentially express CTA and are therefore susceptible to killing by CTA specific T-cells. In addition, CTA transcription is partially regulated by methylation, and therefore demethylating agents maybe used to enhance CTA expression in low expressing tumors and increase susceptibility of these tumor cells to CTA-specific T-cell killings. Our hypothesis is supported by our preliminary data that myeloma stem cells from human myeloma cell line, RPMI 8226, have higher expression of several CTAs, including PRAME, CT7 and NY-ESO1, than the mature myeloma cells (Section C.1). Furthermore, we have already generated PRAME specific cytotoxic T- cells (CTLs) that can kill PRAME positive myeloma cells. In aim 1 we will analyze a large panel of myeloma tumor samples to determine if CTA expression is preserved and/or enriched in primary myeloma stem cells. In aim 2 we will determine if CTLs directed to CTA can directly kill myeloma stem cells using a murine myeloma model. PUBLIC HEALTH RELEVANCE: The long-term objective of this project is to improve the outcome of multiple myeloma (MM), a currently incurable common hematological cancer of plasma cell origin, by targeting the cancer stem cell which play a key role in tumorigenesis and disease relapse using cytotoxic T-cells. The primary goal of this proposal is to generate pre- clinical data for using cytotoxic T-cells to target myeloma stem cells. The fundamental models established and knowledge obtained from this proposal will represent the key for the future design of novel methods toward the long-term cure of myeloma.